Mutagenesis, Tumorigenesis and Carcinogenesis
Today's understanding of mutagenesis of human and animal cells that result in the formation of tumor (tumorigenesis) and cancer (carcinogenesis) is still far from complete. Two opposing hypotheses of the origin of tumor/cancer have existed for many decades. Although old and new hypotheses and observations coexist, in vitro and in vivo studies have not always been consistent, and there seems to be no clear resolution of how tumors/cancers really occur and what roles the genetic mutagenesis and the epigenetic alterations play in the origin and formation of tumors/cancers and how environmental factors affect the respective processes.
The tumorigenic/carcinogenic process in humans and animals appears to involve interactive roles of both genetic factors (tumor suppressor genes, proto-oncogenes, other genes) that direct cell behavior, errors in one or more of the various stages of DNA replication and the like and epigenetic factors that alter cell behavior by modifying on DNA “decorations” and change chromatin “structures”. On top of these complex interplays between genetic and epigenetic factors in the origin and formation of tumors/cancers environmental factors (different physical or cultural environments, X-rays, ultraviolet radiation, mutagenic pollutants, tumor- or cancer-promoting chemicals and environmental toxicants, gender (hormones), diet and the like) also influence the tumorigenesis/carcinogenesis during different stages of life development (maternal/embryonic, neonatal, adolescent, adult and senescent/geriatric). Thus, there is a huge need for further advance our knowledge on mutagenesis, tumorigenesis and carcinogenesis.
Tumor/Cancer Therapies
Current therapies and drugs for treating tumor/cancer have significant difficulties and are, and have been, relatively ineffective. Many tumors and cancers are not treated successfully. This may be because various drug therapies do not properly target the source of the tumor or the cancer, or that they have multi-drug resistance (due to a presence of one or more drug-resistant genes in the genomes of tumor/cancer cells). Also, various genes present in cancer cells and tumor cells are able to be turned “on” or “off” Moreover, there are many different types of tumor/cancer cell lines. Additionally, cells within a tumor/cancer are heterogeneous, rather than homogeneous, in nature. These cells represent a wide variety of genotypic and phenotypic characteristics. Further, no two tumors/cancers of a given origin in either an animal or a human being would contain tumor/cancer cells that are uniformly genetically and phenotypically identical. Moreover, as a tumor/cancer grows, the microenvironment within the tumor/cancer changes, which is caused by decreasing oxygen tension and blood nutrient availability. The tumor/cancer can also be influenced by the environmental factors that are described above, such as hormones and diet. Further, some tumor/cancer cells express drug-resistant genes. Still further, some anti-tumor/anti-cancer drugs, such as thalidomide, cause serious side effects, such as inducing birth defects that would not otherwise occur, causing a new type of tumor/cancer or a toxicity in a patient and/or causing a “stress response” that functions to protect, rather than destroy, cancer cells. Many of the foregoing explanations also explain discrepant or contrasting results that are often obtained from an experimental testing of a potential anti-tumor/anti-cancer drug in vitro as compared with in vivo. For example, typical cell cultures have a high-level of oxygen content as a result of both the culture media and the atmosphere, whereas tumors/cancers growing in an animal or human are typically not exposed to such oxygen-containing factors.
Medical Needs
There has been, and remains, a very significant long-felt, and unresolved, need in the tumor/cancer field for identifying and developing reliable broad-spectrum anti-tumor/cancer drugs and agents that: (i) are effective in killing a variety of tumor/cancer cells; (ii) do not kill or harm normal cells or tissues, which may be adjacent to tumor/cancer cells, the result of which could lead to the death of a patient; and (iii) do not have harmful, uncomfortable or other disadvantageous side effects. Presently existing anti-tumor/cancer drugs and agents typically do not possess each of these three characteristics, and tumor/cancer researchers have tried, and failed, to identify and develop such drugs and agents, which is very challenging for the reasons that are set forth above. Additionally, improper hypotheses, logic and approaches at the cellular level may have been employed by cancer researchers in an attempt to identify and develop such drugs and agents.
Shortcomings of Current Anti-Tumor/Anti-Cancer Drugs
Many conventional anti-neoplastic agents or anti-cancer drugs suffer from drawbacks of severe side effects due to their inhibition on some common processes shared by normal cells [1-3]. On the other hand, highly-specific anti-neoplasm drugs that target at the specific “leaf” level genetic mutation unique to different neoplastic cells are not only too narrow spectrum in their anti-neoplastic effect, but also suffer from losing effect as surviving neoplastic cells can develop resistance by using alternative routes [4-5]. More ironically, some anti-cancer agents can kill the targeted original cancer cells but, in the meantime, lead to the formation of new cancer in the patients [6-8]. The sad reality is that most anticancer drugs [9-10] really do not offer much meaningful benefit to patients' quality of life because they add just an extra week or two of suffering time to the patients' lifespan [11-12].
It turns out that the past as well as the current research on tumors and cancers have been focused too much on the distinct types of tumors and cancers in efforts for identifying their unique genetic mutations and thus develop highly specific anti-tumor/anti-cancer therapy. However, these scattered peripheral “leaf”-level hunting efforts for anti-tumor/anti-cancer drugs often meet with disappointments because many tumors/cancers have the capability of by-passing the very specific route targeted by those narrow-spectrum anti-tumor/anti-cancer drugs specifically designed for a “leaf”-level mutation. Therefore, there has been, and remains, a very significant long-felt, and unresolved, need in the tumor/cancer field for methods for reliably identifying broad-spectrum anti-tumor/cancer drugs that will target on the “root”-level process of tumors/cancers and thus cannot be by-passed by any tumor/cancer cell.
Neoplastic Metabolism: The Root-Process for all Neoplasms Including Tumors and Cancers
The “root” of neoplasia or the “Achilles' heel” of neoplasm, which includes tumor and cancer, resides in the unique metabolic properties and the living processes of the neoplastic cells.
A hallmark of all neoplasms is their oncogenic metabolism, such as a high rate of glycolysis, even under a high oxygen concentration. This phenomenon has been known as the “Warburg Effect” since the 1930s, but remains poorly understood even today [14]. During aerobic glycolysis, pyruvate generated from glucose is not transported into mitochondria for total oxidation for yielding more energy but, rather, is converted to lactate in cytosol and then is excreted outside the cell [15]. For a long time, it was unknown why neoplastic cells would “waste” glucose and choose an energy-“inefficient” metabolism. However, the finding of a “neoplastic or pathological Cori cycle” in which the excreted lactate is carried by the blood to the liver and converted to glucose for reuse by the neoplasm may shed some light in understanding Cachexia, a condition exists in neoplastic patients who suffer massive loss of normal body mass as the neoplasm continues its growth [16]. It turns out that, by avoiding a complete “burn” of glucose to CO2, neoplastic cells preserved some key carbon “skeleton” for neoplastic anabolism which represents another aspect of the oncogenic metabolism. The combined result of the “Warburg Effect” (the aerobic glycolysis) and the pathological “Cori Cycle” (the neoplastic anabolism) thus causes a metabolic imbalance: shifting resource toward neoplastic cells and away from normal cells. This metabolic imbalance ultimately results in systematic failure of patients suffering from a neoplastic disease and their death.
In addition, recent studies have shown that some of the molecular mechanisms underlying the neoplastic metabolism also influence invasion and migration of malignant neoplastic cells which are responsible for metastasis and a wider range of neoplastic diseases [17].
Needs for “Root”-Killing Broad-Spectrum Anti-Neoplastic Agents
Despite great progresses in understanding its etiology and pathology of neoplasia, neoplasm especially the malignant neoplasm such as cancer, still remains as a great risk to human life. Thus, an urgent need in combating neoplasia is to obtain some “root killers” for neoplastic cells [13]. In other words, we need to find wide-spectrum anti-neoplastic agents that are harmful for a variety of tumor/cancer cells but not harmful for normal or healthy cells.
Targeting Neoplastic Metabolism as the Achilles' Heel of Neoplasm
Neoplastic metabolism or tumorigenic/oncogenic metabolism is a common process that all tumor/cancer cells must rely on for their abnormal growth and excessive reproduction (multiplication or proliferation). The neoplastic metabolism or tumorigenic/oncogenic metabolism includes elevated aerobic glycolysis which is known as the Warburg Effect and enhanced neoplastic anabolism which is recognized as a pathological Cori Cycle. Thus, a very effective and much needed approach for treating neoplasia should be based on inhibiting aerobic glycolysis (Warburg Effect), neoplastic anabolism (pathological Cori Cycle), or both at the same time. This approach should yield therapeutic schemes that present much less (fewer) side effects than those associated with conventional anti-cell cycle (reproduction)-based therapy, such as “chemotherapy” or “radiotherapy.” This approach should also yield much broader spectrum anti-neoplastic drugs than those approaches targeting at some rare mutations specific for only a certain type of neoplastic cells. Understanding unique metabolism common to all, or at least most, neoplastic cells and finding agents inhibiting such neoplastic metabolism may hit the Achilles' heel of neoplasm and eradicate neoplasm from its root.
The Shortage and Shortcomings of Primary Neoplastic Cells and their Cell Lines
In addition to the still lacking full translation of the existing insights into the causes of tumor/cancer from bench to bed, a major obstacle in searching for anti-neoplastic agents is ironically the shortage of neoplastic cells suitable for laboratory drug screening [18]. Despite the high incidence of neoplasia, neoplastic cells preserved for research use are relatively few. Collecting primary neoplastic cells is a complex procedure impeded with many red tapes, not to say the great investments and efforts required for characterizing them for research use. Consequently, only a limited number of primary cancer cells have been established as useful cell lines. Thus, there is a need to find more model neoplastic cells for utilization in screening anti-neoplastic agents.
In addition to this limitation in quantity, some neoplastic cell lines passed many times in the laboratories might have accumulated additional mutations that are atypical for natural neoplasm [18]. Thus, it is not uncommon that drugs effective against laboratory lines of cancer cells actually failed dramatically in clinical trials [19]. Some of the anti-cancer drugs actually lead to opposite effects [20-21]. It is also known that some conventional “chemotherapy” sometimes induces tumor regression while simultaneously elicits stress responses that protect subsets of tumor cells [22]. Thus, there is a need for finding more appropriate model neoplastic cells that are suitable for a reliable screening of anti-neoplastic agents.
In view of the above, it would be extremely beneficial to provide cost-effective and reliable methods for successfully discovering, identifying and/or screening for broad-spectrum (or other) anti-neoplastic agents, compounds and/or drugs that are safe for use by human beings and/or animals and may potentially have a therapeutic activity therein, such as partially or fully inhibiting a neoplastic activity or capability of neoplastic cells that are present in such human beings and/or animals, with minimal or no side effects.
Induced Pluripotent Stem Cells
Recently, cells known as induced pluripotent stem (iPS) cells (iPSCs) have been generated in large quantity and diversity [23-25]. The prototype iPSCs were first reported by Yamanaka's team in 2006 for mice [23] and in 2007 for human [24]. These iPSCs were claimed to be induced from differentiated cells by ectopic expressing exogenous transcription factors Oct4, Sox2, Klf4 and c-Myc [23-24] (abbreviated as OSKM) [123] and later known as Yamanaka factors [124]. Another group of iPSCs were reported in 2007 using Oct4, Sox2, Nanog, and LIN28 as the reprogramming factors [25] (abbreviated as OSNL) [123] and later known as Thomson factors [124].
These iPSCs have been described by others as “indistinguishable” from “embryonic stem cells” (ESCs) [26-28] and, thus, have been perceived as “ethical” and “safe” “replacements” of ESCs for cell therapy [29-31], and even for regenerative medicine [32-35].
Discovery: iPSCs are Man-Made Cancer Cells Due to Metabolic Mutation by iPS Reprogramming
It is a discovery of the present inventor that iPSCs are incorrectly programmed stem cells (still abbreviated as iPSCs) or, in other words, man-made cancer stem cells (mmCSCs) [36-38]. Thus, iPSCs can be used as “replacements” for “naturally-occurring cancer cells” in screening agents against neoplastic cells.
It is a further discovery of the present inventor that iPS reprogramming can be linked with neoplastic aerobic glycolysis and anabolism [39]. Thus, iPSCs can be used as model cells for screening agents that specifically inhibit some aerobic glycolysis and anabolism characteristic for neoplasia.
In addition, it is a discovery of the present inventor that iPSCs may possess invasion and migration capability common to malignant neoplastic cells and, thus, may also be used for screening agents against the invasion, migration and metastasis of neoplastic cells.
Invention: Using iPSCs for Screening Anti-Neoplastic Agents for Broad-Spectrum Anti-Tumor/Anti-Cancer Drugs
Based on the discovery of induced pluripotent stem cells (iPSCs) as man-made cancer cells an invention was made that iPSCs can be used for screening anti-neoplastic agents, compounds and drugs that are effective in inhibiting metabolic mutation which serves as a root for neoplastic transformation of normal cells into tumor/cancer cells.
This “odd” invention is apparently against the current mainstream thinking which regards iPSCs as “ethical” replacements for ESCs and “cancer-free” and, thus, even “safe” for cell therapy and regenerative medicine. However, it is right from those reports making “cancer-free” claims for iPSCs that the inventor of this patent application found evidence of the cancer risk for iPSCs [48]. With more detailed reasoning disclosed here for linking iPS reprogramming with neoplastic transformation, iPS researchers should come to a reality that their iPSCs may find a better utility: serving as serendipitous cancer cells for screening anti-neoplastic agents.
The Nonobvious Nature of the Invention
It should be pointed out that the present invention of using iPSCs for screening anti-neoplastic agents is fundamentally different from those inventions of using iPSCs as non-cancerous cells for modeling other diseases [40-41] and screening drugs against those diseases [42-43]. As a matter of fact, iPS researchers have been focused on inventing methods for making iPSCs [44] because human iPSCs have been perceived as “less complicated” “human pluripotent cells” than “embryonic stem cells” (ESCs) and thus are “potentially useful in therapeutic applications in regenerative medicine” [44]. More significantly, claims of generating “cancer”-free “safe” iPSCs suitable for clinical applications are being made repeatedly [28, 45-47], despite the criticisms against the hype contained in these claims [36, 48]. A very recent publication [49] describes acquisition of iPSCs by selecting those cells with transgenes integrated into the so-called “safe harbors,” the genomic regions outside positions known for integration mutation. Even though the iPSCs are still made with the already known oncogenes, a claim of “out of harm's way” was still made [50]. This demonstrates the lack of understanding with regard to how iPS reprogramming results in neoplastic transformation and, thus, how similar iPSCs are to cancer cells than to ESCs.
It should be further pointed out that, even after the publication (on Aug. 2, 2012) of the parent patent application [125] of this patent application, the mainstream of iPSCs research still overlooked the discovery of epigenetic-level metabolic changes or metabolic mutations as the basis for the neoplastic nature of iPS reprogramming and the cancerous nature of the iPSCs. Most researchers are still hoping of creating “safe” iPSCs by eliminating genome-integration of the exogenous genes and removing integration remains of virus-vehicle [126-128]. New claims of creating “cancer-free” iPSCs or even “safer” iPSCs are continuously made [129-130]. At present time. mainstream iPS researchers and top journals are still rejecting the discovery of iPSCs as man-made cancer stem cells (mmCSCs) [37-38] which identifies some cancer risks for various “cancer-free” iPSCs [23-24, 26, 51-53]. Strong efforts are still being made in promoting iPSCs as “ethical” and “safe” ESC replacements for cell therapy and regenerative medicine [28, 54-58]. A recent publication reporting generating iPSCs from dermal fibroblasts of a patient suffering from Hutchinson-Gilford Progeria syndrome (HGPS) [59] has even been regarded as to “lead to novel insights into mechanisms of aging” [60], even though these HGPS-iPSCs are merely some cancerous cells carrying the mutations for HGPS.
Thus, even though some recent publications have noticed the “similarity” between iPSCs and cancer cells [61-62], or the common path between the generation of iPSCs and CSCs [63], the authors of these publications are still contributing the intriguing “parallel” as a result of partial [28] or incomplete [47] reprogramming. At the end, the intrinsic cancer risk of iPSCs has been neglected even in the “comprehensive review [28] or “straight talk” [47] by the leading iPS researchers. Arguments have also been made that “although there are common pathways activated during reprogramming and tumorigenesis, there are fundamental differences between iPS and transformed cancer stem cells” [63].
The awarding of a Nobel Prize in Physiology or Medicine to Yamanaka [131] is a strong indication that even the top scientists in the world still lack a minimal appreciation for the discovery of iPSCs as “man-made cancer stem cells” (mm-CSCs) [36-39, 125], especially when this discovery was presented to the Nobel Prize selection committee [132]. Thus, it would not have been obvious at the time the present invention was made that iPSCs could be used for screening anti-cancer drugs when they are still considered as safe “replacements” of ESCs for cell therapy and even for regenerative medicine [133].
The non-obviousness of the deep insight on the intrinsic oncogenic and neoplastic transformation enabled with the iPS reprogramming is further evidenced by the rejection of the mainstream against the publication of the unique view by the inventor (see note in [39]) and the neglect of the prior art disclosed in the published patent application [125] by the mainstream. Some researchers in the field still tried to distinguish iPS reprogramming from oncogenesis and neoplastic transformation [134]. The fact is, there is no report of using iPSCs for screening anti-cancer drugs even after the publication of the patent application disclosing the invention of using iPSCs for screening anti-neoplastic agents, despite the strong need for developing anti-cancer drugs. Thus, even as today, the invention of using iPSCs for screening anti-neoplastic agents remains non-obvious even to the leading scientists in the related technical fields.
For example, a recent review published in Stem Cells and Development, which published a critical review on iPS cells in 2008, but later in 2009 rejected the submission of a manuscript describing cancer cell formation by iPS reprogramming (see note in [39]), considers “induced pluripotency and oncogenic transformation are related processes” [135]. It emphasizes more the “distinctions” between induced pluripotent stem cells (iPSCs) and oncogenic foci (OF) by stating that “iPSCs and OF shared a limited number of genes that were upregulated relative to parental fibroblasts” and “iPSCs and OF were distinct in that only iPSCs activated a host of pluripotency-related genes, while OF activated cellular damage and specific metabolic pathways” [135]. This review concludes that “OF and iPSCs are related, yet distinct cell types, and in which induced pluripotency and induced tumorigenesis are similar processes” [135].
The above view of treating iPSCs and OF as distinct cell types and separating iPS reprogramming from oncogenic transformation is incorrect because some of the differences between OF and iPSCs may be caused by the heterogeneous nature of OF due to the inherent variations in the differentiation status of the composing cells and the high purity of iPSCs as they are selected out based on some stemness markers [36]. However, that difference should not rule out the same oncogenic tumorigenesis or neoplastic transformation pathway used for forming OF and iPSCs. Indeed, using Nanog, a stemness marker and also a reprogramming factor used in some iPS reprogramming, reprogrammed oncogenic foci (ROF) with iPSC-like cells have been generated [135].
Evidences Supporting the Discovery and the Invention
Nevertheless, it is at least admitted by Yamanaka that iPSCs generated with his patented method have undeniable cancer risk as he recognized in his correction [137] to his Science publication [138] after thanking criticisms made by the inventor [139] of this invention [125]. Such risk was also confirmed by many later studies, including Yamanaka's own studies [87-89] The cancer risk may still exist for iPSCs generated with integration-free reprogramming methods [140, 141]. More convincingly, a Letter-to-the-Editor published in Stem cells showed that human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection [142].
Interestingly, the unavoidable cancer risk associated with the prototype reprogramming method [136] has forced Yamanaka to file additional patent applications to claim “inventions” of selecting “safe” iPSCs [143,144]. However, these “inventions” are obvious responses to the public criticisms [36-39, 90-92, 125, 139, 141, 142, 168] made by the inventor of this patent application and actual implementations of some suggestions made in those public criticisms.
After all, the claims made in these patent applications may be invalid as the methods disclosed really lack any effective measure to overcome the intrinsic neoplastic nature of the iPS reprogramming and, thus, are really incapable of selecting cancer-free safe iPSCs [145].
Oncogenic Metabolism Resulted from Epigenetic Changes via iPS Reprogramming
The “root” cause for the neoplastic transformation via the iPS reprogramming is actually not the mutagenesis conventionally associated with random integration of exogenous genes and their vectors. It is some specific epigenomic alterations associated with the elevation of some specific reprogramming factors, as a result of introducing their genes, proteins, or RNAs, or even their metabolites or similar chemicals, into the recipient cells. The reprogramming factors have potent capability of activating the cell growth and reproduction of the recipient cells and, thus, make them highly proliferative. When such rapidly multiplying cells were selected out based upon some stemness markers, the so-called “induced pluripotent stem cells” (iPSCs) are obtained [36-39, 90-92, 125, 139, 141, 142, 168].
However, by clearly identifying the various linkages between iPS reprogramming and neoplastic transformation, it is hoped that the present application will establish a solid foundation for arguing iPSCs as a kind of “neoplastic” cells very similar to “natural” cancer cells and, thus, justifying their use as serendipitous “replacements” for cancer cells in methods for screening agents against neoplastic cells.
c-Myc and Lin28
It turns out that Myc (c-Myc), a very important iPS reprogramming factor, is a notorious oncogene and a master transcription factor that integrates cell proliferation with metabolism through its regulation of thousands of genes including microRNAs (miRNA) [64]. In addition to its known function in regulating the cell cycle and glucose metabolism [65], Myc also stimulates glutamine catabolism [66] through the repression of miRNAs miR-23a and miR-23b [67].
More significantly c-Myc enhances the expression of poly-pyrimidine tract binding protein PTB (also known as hnENPI), hnRNA1 and hnRNA2, and leads to selective expression of pyruvate kinase isoform 2 (PKM2) [65]. PKM2 is the M2 splice isoform of pyruvate kinase (PK) [68] which is a key enzyme for aerobic glycolysis [69], as compared with the M1 splice isoform of pyruvate kinase (PKM1) which is a key enzyme for oxidative phosphorylation. Thus, the selective expression of specific isoform of PK serves as a toggle switch for shifting mass-energy metabolism between an energy production-efficient oxidative phosphorylation and a mass production-efficient aerobic glycolysis.
It is interesting to notice that PKM2 is the dominant form of PK in “embryonic cells” and PKM1 is the dominant form of PK in the “adult cells” [68-69]. This age-specific expression of different isoforms of the same enzyme reflects the physiological need as PKM2 is needed for glycolytic anabolism supporting mass increase in the growth stage of the life, and PKM1 is needed for allowing established cells to perform more energy-consuming functions at the grown up stage of life. Thus, a change in the expression of different isoforms of the same enzyme leads to different modes of metabolism in different life stages. Overgrowth of cell mass, such as neoplasm, is not a desired shift.
Unfortunately, the re-expression of PKM2 [70] activates those “resting” cells and drives them from normally a “quiescent” state into a “hyper-proliferation” state [71]. This adulthood expression of an embryonic enzyme isoform does not lead to the “rejuvenation” of the whole organism, but a formation of some harmful, and even deadly, neoplasm.
More than just contributing to the transformation of “normal cells” into “neoplastic cells,” c-Myc coordinately regulates the expression of 13 different “poor-outcome” cancer signatures [17]. In addition, functional inactivation of c-Myc in human breast cancer cells specifically inhibits distance metastasis in vivo and invasive behavior in vitro [17]. So, c-Myc may also contribute to the acquisition of metastatic capability of the “neoplastic cells.”
Therefore, iPSCs generated with inducing factors including c-Myc may naturally possess some basic neoplastic features known to natural cancer cells.
The iPSCs generated without c-Myc may also possess oncogenic nature. For example, Lin28, an inducing factor used in place of c-Myc [25], has recently been found in association with cancers [72-74]. More importantly, Lin28 has been shown as a Myc-downstream factor exerting the similar effect as Myc [74-75].
iPS Reprogramming Factors and Cancer Risk
As a matter of fact, iPS reprogramming factors currently employed are more or less associated with various cancers [37-38, 76-77]. Thus, the oncogenic potential is intrinsic for iPS reprogramming, at least for the proto-type iPS reprogramming methods [39]. This intrinsic oncogenic potential is intensified when a tumor-suppressing mechanism is inhibited or knocked out [39]. Unfortunately, many iPS researchers just do not want to face this dark side of iPS reprogramming, and continue at looking at the “bright” side of their discoveries [78]. They emphasize the enhanced “efficiency” of iPS reprogramming and elevated yield of the iPSCs by knocking out the tumor-suppressing mechanisms [79-83] while ignoring the increased risks of cancer potential from these tumor suppression mechanism-jeopardized iPSCs [84-85].
Nevertheless, increasing reports are presenting observations of chromosomal aberrations [86] and cancer-related epigenome changes [87, 146] in iPSCs. There are also reports documenting formation of rhabdomyosarcomas iPSCs [88]. These observations have led to some concerns over the “variation” in the safety of iPSCs [89]. But, claims of generating “transformation-deficient” [45] and, thus, “safe-induced pluripotent stem cells” (safe-iPSCs) with therapeutic potential [46] are still being made. It has been believed that “although there are common pathways activated during reprogramming and tumorigenesis, pluripotent stem cells and tumorigenic cells have important differences” and thus the critical distinctions between true cancer cells and reprogrammed somatic cells may be that reprogrammed cells remain genetically intact” [61]. Thus, despite a clear message arguing the intrinsic distinctions between iPSCs and ESCs [90-92] and some later experimental reports supporting this argument [93-94], leading iPS researchers still reject the intrinsic distinctions between iPSCs and ESCs and the high similarity between iPSCs and cancer cells [95]. The most recent review on iPSCs still claims: “Numerous studies indicate that, at least for some clones, iPSCs are similar if not indistinguishable from ESCs derived from embryo or nuclear transfer experiments” and “somatic cells can be reprogrammed to a pluripotent state, which is molecularly and biologically indistinguishable from that of ESCs” [28] and a straight talk states that there's no reason . . . to think that true bona fide iPSCs cannot function as well as ESCs [47]. This attitude is also reflected by the lack of appreciation of the cancerous nature of iPSCs even by experienced stem cell researchers.
Amazingly, even some cancer researchers apparently still lack an understanding of the cancerous nature of the iPSCs. A recent study has found “a Myc network accounts for similarities between embryonic stem and cancer cell transcription programs” [96]. Even though some iPSCs were also included in this study, the report failed in identifying iPSCs as cancer cells. As a matter of fact, the corresponding author of this report did not even answer the straight question from the present inventor on whether or not iPSCs are cancer cells.
However, it is hoped that, continued dissection of the iPS reprogramming process may not only lead to a comprehensive identification of a sufficient factor set for complete and safe somatic to pluripotent reprogramming [97] but also an increased awareness of the neoplastic nature of iPS reprogramming [39] [125]. More importantly, if the application of this invention is placed into practice, the cancerous nature of iPSCs may be made very apparent, when anti-neoplastic agents, compounds and/or drugs discovered via methods disclosed in this application are also very effective in killing “natural cancer cells.”
Genetic and Epigenetic Mutations affecting the Warburg Effect
It is important to point out that iPS reprogramming can turn “normal cells” neoplastic even without any genetic modification and/or a cell reproduction event. This feature will be a key point for the present invention which is focused on discovering anti-neoplastic agents, compounds and drugs that are effective in inhibiting the metabolic mutation serving as a root process for supporting the malicious competition of “neoplastic cells” against “normal cells.” In the past, cancer research has been heavily focused on genetic mutations, including mutations in mitochondria, as causes for neoplasia [99-100]. The discovery of Warburg Effect even led some researchers to believe that neoplastic cells have abnormal mitochondria. The outcome of this genetic cancer dogma is the focus of searching anti-cancer drugs that fix the genetic mutations, including mitochondria mutations [101]. However, many drugs targeting the effects of genetic mutations often fail in killing tumor cells, and even succeed in killing normal cells [102].
It turns out that, many times, it is the mitochondrial uncoupling, the abrogation of ATP synthesis by mitochondria, promotes the Warburg Effect in some neoplastic cells and contributes to their resistance to chemotherapy targeting mitochondria [103]. These cancer cells may shift to the oxidation of non-glucose carbon sources to maintain mitochondrial integrity and function [103]. More importantly, increased level of c-Myc in cancer cells causes an increase in level of glutaminase, a protein that helps cells convert amino acid glutamine into an energy source. The breakdown of glutamine provides cancer cells with a carbon source. In fact, glutamine can serve as a major nutrient for cancer cells [104], especially when facing glucose deprivation [105]. Also worth of notice is that mutation in some genes such as KRAS or BRAF often lead to up-regulation of the expression of GLUT1 (encoding glucose transporter-1) and SGLT1 [106]. Thus neoplastic cells often have enhanced glucose uptake and glycolysis, and can survive even at low glucose concentration [107]. Thus, neoplastic cells may still have normal mitochondria despite their abnormal use. Amazingly, drugs targeting mitochondria sometimes kill normal cells more effectively, but exert less or even no harm to neoplastic cells, which use less or even shut down their mitochondria.
Metabolic Mutation/Alteration, Metabolism Switch, and Oncogenic Metabolism
A metabolic mutation or a metabolism switch may be a predominant feature in cancer cell formation. This change may happen at the epigenetic levels and this oncogenic metabolism has attracted more and more research attention recently. A simple RNA splicing which is a modification of an RNA transcript through removing of introns and joining of exons may produce different proteins out of the same gene [108-109]. This epigenetic regulation plays a very important role in normal development as well as neoplastic tumorigenesis [110-111]. Very often, the alternative splicing changes the mode of mass/energy metabolism [112-113] and this alteration in splicing can be influenced by the conditions in which the cells reside [114-115]. A very recent study just confirmed that some cancer-related epigenome changes have been found in iPSCs [87].
It should be pointed out that, although epigenetic changes have been recognized in iPS reprogramming [146], it was not until recently that mainstream journals formally recognized the “shared mechanisms” between induced pluripotency and oncogenesis [147-148]. However, these examinations often focus on the “parallels” between iPS reprogramming and cancer epigenetics in terms of the common transcription factors (TFs) used in the (iPS) “reprogramming” and the (oncogenic)“transformation” [148] but generally failed to realize the common metabolic transformation between iPS reprogramming and neoplastic transformation as discovered by the inventor of this patent application.
Evidence for Metabolic Mutation or Oncogenic Metabolism in iPSCs
Studies have shown that oncogenes such as Myc [116-117] play very important roles in contributing to glycolytic metabolism in cancer cells [70]. Studies also show that glucose deprivation induces oncogenic mutations and thus a switch to oncogenic metabolism [107]. The fact that hypoxia enhances the generation of iPSCs [118] indicates that iPSCs may have switched into a neoplastic glycolysis.
Two reports showed that butyrate promoted generation of iPSCs [149-150]. However, the promotion of iPSCs generation by butyrate was shown as c-Myc-dependent in one study [149] while the other study showed remarkable butyrate stimulation on reprogramming in the absence of KLF4 or MYC transgene [149]. The later study showed enhanced histone H acetylation, promoter DNA demethylation, and expression of endogenous pluripotency-associated gene [150].
To further support the validity of the present invention of using iPSCs for screening anti-neoplastic agents or anti-cancer drugs some additional experimental studies related to the epigenetic landscape of iPSCs [151], especially in comparison to the metabolism of cancer cells [179, 180], are described herein.
On Aug. 3, 2011, a research paper entitled “somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming” was published [152]. This study reported that “[T]emporal sampling demonstrated glycolytic gene potentiation prior to induction of pluripotent markers. Functional metamorphosis of somatic oxidative phosphorylation into acquired pluripotent glycolytic metabolism conformed to an embryonic-like archetype. Stimulation of glycolysis promoted, while blockade of glycolytic enzyme activity blunted, reprogramming efficiency. Metaboproteomics resolved upregulated glycolytic enzymes and downregulated electron transport chain complex I subunits underlying cell fate determination. Thus, the energetic infrastructure of somatic cells transitions into a required glycolytic metabotype to fuel induction of pluripotency.” Thus, this study provided a supporting evidence for the theoretic and also logical discovery of glycolysis as a basis for the neoplastic transformation via iPS reprogramming [39, 125].
In 2012, another research article showed metabolic changes from an oxidative state to a glycolytic state during iPS reprogramming and also identified metabolites that differ between iPSCs and ESCs [153]. Interestingly, this study used an Extracellular Flux analyzer manufactured by Seahorse Bioscience (Massachusetts, United States) to measure the basal oxygen consumption rate (OCR) as a measure of mitochondrial respiration and the extracellular acidification rate (ECAR) as a measure of glycolysis-[125] More interestingly, by comparing the effects of some chemicals on the reprogramming efficiency, this study showed that a glycolysis inhibitor, 2-deoxy-D-glucose (2-DG), inhibited the reprogramming efficiency while a glycolysis stimulator, D-fructose-6-phosphate (F6P), increased reprogramming efficiency [153]. These results showed that, by using iPSCs and by measuring the effects of agents on the metabolism associated with neoplastic transformation, anti-neoplastic agents or anti-cancer drugs can be effectively identified. Unfortunately, even though the authors of this study have stated that their “study not only highlights the importance of metabolism in inducing pluripotency, but suggests that understanding the metabolic changes associated with somatic cell reprogramming may also shed light on the metabolic mechanisms regulating cancer” [153], they did not recognize iPSCs as cancer cells.